18 (1), 2015 l 31-38 DOI: 10.1515/bjmg-2015-0003

ORIGINAL ARTICLE

INVESTIGATION OF FASCICULATION AND ELONGATION ζ-1 (FEZ1) IN PERIPHERAL BLOOD REVEALS DIFFERENCES IN EXPRESSION IN PATIENTS WITH SCHIZOPHRENIA Vachev TI1,2, Stoyanova VK2,*, Ivanov HY2, Minkov IN1, Popov NT3

*Corresponding Author: Associate Professor Vili K. Stoyanova, M.D., Ph.D., Department of Pediatrics and Medical Genetics, Medical University ‒ Plovdiv, 15A Vasil Aprilov St., 4000 Plovdiv, Bulgaria. Tel: +359-32- 602-431; Fax: +359-32-602-593. E-mail: [email protected]

ABSTRACT peripheral blood of patients with schizophrenia. Our results suggested a possible functional role of FEZ1 Schizophrenia (SZ) is a chronic neuropsychiatric in the pathogenesis of schizophrenia and confirmed disorder characterized by affective, neuromorpho- the utility of peripheral blood samples for molecular logical and cognitive impairment, deteriorated social profiling of psychiatric disorders including schizo- functioning and psychosis with underlying molecular phrenia. The current study describes FEZ1 gene ex- abnormalities, including gene expression changes. pression changes in peripheral blood of patients with Observations have suggested that fasciculation and schizophrenia with significantly down-regulation of elongation protein ζ-1 (FEZ1) may be implicated FEZ1 mRNA. Thus, our results provide support for in the pathogenesis of schizophrenia. Nevertheless, a model of SZ pathogenesis that includes the effects our current knowledge of the expression of FEZ1 in of FEZ1 expression. peripheral blood of schizophrenia patients remains Keywords: Fasciculation and elongation protein unclear. The purpose of this study was to identify ζ-1 (FEZ1); gene expression; quantitative reverse- the characteristic gene expression patterns of FEZ1 transcriptase polymerase chain reaction (qRT-PCR); in peripheral blood samples from schizophrenia pa- schizophrenia (SZ) tients. We performed quantitative reverse-transcrip- tase (qRT-PCR) analysis using peripheral blood from INTRODUCTION drug-free schizophrenia patients (n = 29) and age and gender-matched general population controls (n = 24). Schizophrenia (SZ) is a severe mental disorder For the identification of FEZ1 gene expression pat- affecting approximately 1.0% of the human popula- terns, we applied a comparative threshold cycle (CT) tion worldwide [1]. Twin, family and adoption studies method. A statistically significant difference ofFEZ1 strongly support that genetic factors play an important mRNA level was revealed in schizophrenia subjects role in the etiology of SZ. Currently, numerous genetic compared to healthy controls (p = 0.0034). To the studies, including linkage scans and their meta-analy- best of our knowledge, this study is the first describ- ses, candidate gene association analyses, gene expres- ing a down-regulation of FEZ1 gene expression in sion and genome-wide association studies (GWAS) have identified particular and chromosomal loci with the disorder [2-4]. Circulating blood is eas- 1 Department of Plant Physiology and Molecular Biology University ily accessible material and has been suggested as an of Plovdiv “Paisii Hilendarski”, Plovdiv, Bulgaria alternative to tissue samples for molecular profiling of 2 Department of Pediatrics and Medical Genetics, Medical University – Plovdiv, 4000 Plovdiv, Bulgaria human disease and disease risk [3,5]. This is based on 3 State Psychiatry Hospital Pazardzk, Pazardzk, Bulgaria the capacity of peripheral blood to reflect pathological

31 FEZ1 EXPRESSION IN SCHIZOPHRENIA changes in the body. As the brain tissue is not easily International Neuropsychiatric Interviews were done accessible for investigation, blood-based expression by a certified psychiatrist to evaluate the diagnosis profiling is increasingly being undertaken to search of paranoid SZ only, on Diagnostic and Statistical potential biomarkers for SZ [6,7]. Fasciculation and Manual of Mental Disorders (IVth edition) criteria elongation protein ζ-1 (FEZ1) is one of the first identi- and to exclude any mental disorder in the controls. fied binding partners of Disrupted-in Schizophrenia Important inclusion criteria were that the participants 1 (DISC1), a susceptibility gene for major mental had not received any medication (even psychotro- disorders including SZ in yeast two-hybrid screen of pic) 1 month before blood sampling and they had a an adult human brain library [8]. Moreover, proteomic standard breakfast, so they were assessed in a state techniques revealed that FEZ1 protein interacts with of exacerbation. Persons with other chronic medical various intracellular partners, such as motor signal- and current acute somatic/neurologic illness, alcohol ing, and structural , one of which is DISC1 or drug abuse/dependancy were also excluded. The [9]. Currently, the role of FEZ1 in mammalian neuro- sample population included 15 males/14 females di- nal development in vivo is not well understood. The agnosed with SZ and 24 age- and gender-matched FEZ1 null mice exhibit hyperactivity and enhanced general population controls (12 males/12 females) responsiveness to psychostimulants [10], supporting with no evidence for any psychiatric or neurological a po-tential contribution of FEZ1 dysfunction to SZ. disorder in first-grade relatives (Table 1). Recently, additional evidence based on sequencing Table 1. Age descriptive statistics. of DISC1-interacting partner genes including FEZ1 revealed an increased burden of rare missense variants Groups n Mean SD SE Mean in SZ susceptibility in an isolated northern Swedish SZ 29 45.62 12.565 2.333 population [11]. In contrast, the FEZ1 gene shows no Controls 24 46.38 12.673 2.587 association to SZ in Caucasian or African American SD: standard deviation; SE: standard error; SZ: schizophrenia. populations [12]. However, no association was found in either population between specific haplotypes and Blood Collection and RNA Isolation. Blood any of the psychiatric disorders [13]. Interestingly, samples from the patients and control groups were there is a significant reduction of FEZ1 mRNA in collected in PAXgene Blood RNA collection tubes both hippocampus and dorsolateral prefrontal cortex (PreAnalyticX GmbH, Hombrechtikon, Switzerland) of SZ patients and also an association of the DISC1 that contain a reagent that lyses blood cells and im- genotype and FEZ1 mRNA levels [9]. All these find- mediately stabilizes intracellular RNA to preserve the ings raise the possibility that FEZ1 and DISC1 may gene expression profile. To reduce any potential bias assist in regulating both neuronal development and in gene expression due to diurnal variation, blood was risk for SZ. We hypothesized that previously shown drawn in the morning, from all of the subjects. We FEZ1 mRNA levels in prefrontal cortex of SZ patients used the PAXgene Blood miRNA Kit (PreAnalyticX) may be relevant to those in peripheral blood tissue. to extract total RNA from the blood samples [14]. To- The aim of this study was to identify gene expression tal RNA was then quantified by absorbance at A260 patterns of FEZ1 in peripheral blood samples from nm using Epoch Micro-Volume Spectrophotometer SZ patients. System (BioTek, Winooski, VT, USA) and the purity was estimated by the ratio A260/A280 nm. The ab- MATERIALS AND METHODS sorbance ratio of 260 nm and 280 nm (A260/A280) was between 1.93 and 2.1 for all samples included for Ethics Statement. This study and the informed further analysis. The RNA integrity was confirmed consent forms were approved by the Medical Uni- by non denaturing agarose gel electrophoresis, which versity of Plovdiv Ethics Committee. was then stored at ‒80 °C until further analysis. The Participants. Written informed consent was resulting RNA was treated with RNase-free DNase I obtained from 29 patients recruited at the State Psy- (Promega BioSciences, San Luis Obispo, CA, USA) chiatry Hospital Pazardjik, Pazardzk, Pazardzk, Bul- according to the manufacturer’s protocol and checked garia and 24 healthy volunteers. Routine psychiatric for DNA contaminations prior to copy DNA synthesis examination, wide medical hystory and the Mini- step.PAGE

32 BALKAN JOURNAL OF MEDICAL GENETICS Vachev TI, Stoyanova VK, Ivanov HY, Minkov IN, Popov NT

Quantitative Reverse-Transcription Poly- the test sample and the calibrator samples was made merase Chain Reaction (qRT-PCR) Analysis of using the equation:

FEZ1 mRNA Level. The qRT-PCR analyses were Ct(test) = Ct(target, test)‒Ct(reference, test) performed in at least three physically separate rooms Ct(calibrtator) = Ct(target, calibrator)‒Ct(reference, calibrator) in to order to reduce the chance for contamination. Second, normalization of the Ct of the test sam- Copy DNA for the protein coding gene was synthe- ple to the Ct of the calibrator was made using the sized from total RNA with oligo (dT)18 primer using equation:

RevertAid First Strand cDNA Synthesis Kit accord- Ct = Ct(test)‒Ct(calibrator) ing to the assay protocol (Thermo Fisher Scientific, Finally, calculation of the expression ratio with Waltham, MA, USA). Reverse transcription reactions the following equation: contained 1 µg of total RNA samples, 1 µL oligo 2‒Ct = normalized expression ratio.

(dT)18 primer and nuclease free water to a final vol- The result obtained is the fold increase (or de- ume of 12 µL, after incubation at 65 °C for 5 min., crease) of the target gene in the test samples relative we added 4 µL 5X RT buffer, 1 µL RiboLock RNase to the calibrator sample. Inhibitor (20 U/µL) (Thermo Fisher Scientific), 2 Statistical Analyses. All statistical calculations µL 10 mM dNTP Mix and 1 µL RevertAid MMuLV were performed using the Statistical Package for the Reverse Transcriptase (200 U/µL) (Thermo Fisher Social Sciences (SPSS) software, version 20.0 (SPSS Scientific), to final volume of 20µ L. A relative gene Inc., Chicago, IL, USA). Analysis of variance (ANO- expression method was employed to determine gene VA) t-test of Ct was used to examine differences expression levels. The reactions were set up in dupli- in expression levels of FEZ1 mRNA across healthy cate in a 96-well format using the 7500 Real-Time controls and SZ subjects. A Spearman correlation PCR system (Applied Biosystems, Foster City, CA, analysis was also done for searching relation between USA) and Maxima SYBER Green/Rox qRT-PCR Kit expression levels and demographic characteristics of (Thermo Fisher Scientific). Amplification of the single the tested individuals. amplicon coresponding to the FEZ1 sequence was To investigate the characteristics of FEZ1 gene confirmed by monitoring the dissociation curve (melt- expression as potential diagnostic biomarkers in SZ ing curve analysis) and by agarose gel electrophoresis. patients, a ROC (receiver operating characteristic) The qRT-PCR forward and reverse primers for FEZ1 curve was done and AUC (area under the ROC curve) used in this study were 5’-GGG ACT GCA TGA GAC was calculated. Statistical tests were two-sided with CAT GT-3’ and 5’-TTG AGG GCT GTA GCC AGA a p value of 0.05. CT-3’, respectively [15]. B-actin (ACTB) was used as The STRING Pathway Analysis of FEZ1 Pro- an internal control for normalization. ACTB-specific tein Interactions. The FEZ1 interaction data for each qRT-PCR primers were as follows: forward 5’-AGT validated protein was obtained using the STRING da- GTG ACG TGG ACA TCC GCA-3’ and reverse 5’- tabase (version 9.0; http://string-db.org/) (Figure 1). GCC AGG GCA GTG ATC TCC TTC T-3’. After performing the qRT-PCR analysis, the Ct RESULTS values were measured, different methods could be used to determine the expression level of the target In order to identify whether a key DISC1 (in- gene in the test sample relative to the calibrator sam- teracting partner gene) FEZ1 expression is altered ple. Here, we used the Livak and Schmittgen method in SZ patients we performed a detailed analysis of [16], also known as the comparative 2‒Ct method. All peripheral blood samples. To test this, we evaluated the analyzed SZ samples showed mean Ct values FEZ1 mRNA levels in SZ patients. range from 26.7 to 34.9 and mean control Ct values As the data were normally distributed, we per- ranged from 28.0 to 31.3. The mean Ct values of the formed two-tailed t-test. A statistically significant reference ACTB gene for SZ samples ranged from difference of FEZ1 mRNA level was revealed (p = 16.6 to 21.7 and the range for control samples from 0.0068) (Figure 2). Relative quantification (RQ) cal- 16.3 to 18.0, respectively. culations were done using the 2‒Ct method [16]. The First, for the normalization of the Ct values of results clearly demonstrate down-regulation of FEZ1 the target genes to that of the reference gene, for both mRNA levels in SZ patients. The data are presented

33 FEZ1 EXPRESSION IN SCHIZOPHRENIA

A) B)

Figure 1. Network visualization of a key protein partners of FEZ1. A) The STRING screenshot shows an experimentally validated protein partners of FEZ1. B) Protein partners of FEZ1 (shown in red) in GO Biological Processes, term: neurogenesis.

Figure 3. Presentation of the results as fold changes in gene expression normalized to an endogenous reference gene relative to the healthy control patients. Log transformed FEZ1 RQ of individual SZ subjects (in black), where 1 on the y-axis is average expression in controls. The average change of RQ level is shown in gray.

Figure 2. The figure shows the ΔCt values of FEZ1 agreement with the results observed in inter-patient expression across the analyzed SZ patients and healthy variation in the expression studies by other authors. controls compared with the cycle in which they The PCR amplification of the specific products corre- were obtained. The qRT-PCR procedure was used to determine relative quantification levels ofFEZ1 mRNA sponding to ACTB and FEZ1 amplicons was confirmed (RQ FEZ1) in peripheral blood from 29 patient samples. by monitoring the dissociation curve (Figure 4). The Spearman test did not manage to reveal any significant correlation with age and sex in the tested as the fold change in gene expression normalized groups (p = 0.65/p = 0.54). The ROC also displayed to an endogenous reference gene and relative to the good characterization of state prediction with AUC = healthy control patients (Figure 3). 0.728, (p = 0.0011) (Figure 5). All this was consistent The observed differences in individual expres- with a key role of FEZ1 protein as interacting partner sion levels clearly demonstrate the heterogeneity in with DISC1 as SZ susceptibility genes (Figure 1A), the expression profile of the analyzed gene that is in and FEZ1 protein partners involved in neurogenesis

34 BALKAN JOURNAL OF MEDICAL GENETICS Vachev TI, Stoyanova VK, Ivanov HY, Minkov IN, Popov NT

Figure 4. The qRT-PCR data showing a DNA melt profile result for amplification of the specific single product in qRT-PCR analysis. Specific single products corresponding toACTB in SZ and healthy control patients top panel and FEZ1 amplicons bottom panel, respectively, were confirmed by monitoring the dissociation curve (melting curve analysis). The melting dissociation, performed on FEZ1and ACTB, allowed confirmation of the specificity of the amplifications. The melting temperatures of FEZ1 amplicons were 84 °C ± 1 °C, whereas ACTB had a melting temperature of 83 °C ± 1 °C. as shown in (GO) Biological Pro- of which are also involved in neurodevelopmen- cesses (p = 0.063) (Figure 1B). tal processes implicated in the patho-physiology of psychiatric disorders. Experiments with primates DISCUSSION and rodents demonstrate that FEZ1 and DISC1 have overlapping temporal and spatial expression patterns Despite the identification of numerous SZ sus- [17,18]. Both proteins are expressed in the pyramidal ceptibility genes, the pathology of SZ still remains neurons of the developing hippocampus, the cere- unknown. At the molecular level, a large number of bral neocortex and the olfactory bulb. Moreover, potential DISC1 binding partners have been iden- disruption of the DISC1/FEZ1 interaction inhibits tified from a yeast two-hybrid screen [12], many DISC1-stimulated neurite outgrowth in PC12 cells

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obvious limitation of previous expression studies is the use of human postmortem brain tissue for quan- titative analyses of gene expression profile, primarily because postmortem brain tissue from SZ patients is extremely rare and highly prized. Additionally, identification of gene expression profile can be com- plicated by a variety of confounding factors such as pH, drugs, cause of death, etc. In contrast, in attempts to overcome these limitations, we used RNA storage and extraction systems that block and preserve RNA for downstream expression study, so that the expres- sion level that we identified really reflects the current physiological state of the analyzed patients. All these changes in expression levels probably are not due to gene polymorphisms, as such was not detected in a large SZ cohort [19], but can be attributed to various epigenetic mechanisms that alter distinct molecular Figure 5. Performance of the FEZ1 prediction model. pathways. As hypomethylation was observed in the The ROC for FEZ1 expression was performed to evaluate the prediction accuracy. The dotted diagonal line exonic region of HTR2A and MB-COMT promoters in represents random classification accuracy (AUC 0.5). the DNA derived from saliva in SZ [20], it is possible The accuracy of FEZ1 as a single predictor was AUC that epigenetic factors leading to down-regulation 0.728 [95% confidence interval (CI) minimum 0.589/ of FEZ1 in schizophrenic brains may also reflect maximum 0.841; SE (standard error) 0.0698] with 55.2% in peripheral blood and result in the reduction of sensitivity and 87.5% specificity between schizophrenic and healthy controls subjects. the expression in these tissues. However, additional studies applying RNA sequencing analysis for iden- tifying peripheral blood-based biomarkers that could represent brain expression and epigenetic aberrations [8], thus, decreased FEZ1 levels could influence remain a key step in implication of these findings in DISC1-stimulated functions. pathogenesis, diagnosis and future therapy. On the one hand, this study is the first describ- ing a FEZ1 gene expression change in peripheral ACKNOWLEDGMENTS blood of patients with SZ; on the other hand, this change corresponds to the down-regulation in pre- We gratefully thank all the probands and their frontal cortex and hippocampus of schizophrenic families who were enrolled in this study and all the patients [9]. Due to the fact that FEZ1 interacts control subjects who donated blood samples. with DISC1, a susceptibility gene for major men- tal disorders to synergistically regulate dendritic Declaration of Interest. This study was sup- growth of newborn neurons in the adult mouse hip- ported by MU – Plovdiv Project No. HO ‒ 4/2011. pocampus. We assume that any interaction related The authors report no conflicts of interest. The au- to another FEZ1 partner would be compromised thors alone are responsible for the content and writing or at least influenced due to changes in the expres- of this article. sion of the FEZ1 transcript that would change the level of translated protein and its involvement in REFERENCES complexes associated with susceptibility for SZ functions (Figure 1). 1. Perala J, Suvisaari J, Saarni SI, Kuoppasalmi K, Thus, our results provide support for a model Isometsä E, Pirkola S, et al. Lifetime prevalence of SZ pathogenesis that includes the regulatory ef- of psychotic and bipolar I disorders in a general fects on FEZ1 gene expression in peripheral blood population. Arch Gen Psychiatry. 2007; 64(1): specific for patients with exacerbation of SZ. One 19-28.

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